1. Field of the Invention
This invention relates to a novel microbial strain and a method of biodegrading chlorinated organic compounds such as trichloroethylene (TCE) and dichloroethylene (DCE) by using it. It also relates to a method of environmental remediation by treating aqueous media such as drain water and waste aqueous solutions containing chlorinated organic compounds. Such a method is useful for recovering the soil and the air (gaseous phase of the environment) from contamination particularly when the air is contaminated by chlorinated organic compounds.
2. Related Background Art
In recent years, environmental contamination by hardly decomposable chlorinated organic compounds that are harmful to living things has been a major environmental problem. Particularly, it is assumed that the soil of industrial zones relating to paper manufacturing, pulp industry and precision machining has been contaminated to a large extent by chlorinated organic compounds including chlorinated aliphatic hydrocarbon compounds such as tetrachloroethylene (PCE), trichloroethylene (TCE) and dichloroethylene (DCE) and this assumption has been justified by a number of reports on the environment of industrial quarters around the world. The chlorinated organic compounds discharged into soil are then dissolved into underground water and spread to surrounding areas to contaminate them. Most of these compounds are strongly suspected to be carcinogenic and provide a major social problem particularly because many people are relying on underground water for water supply and these compounds are highly stable in the environment.
Under these circumstances, it is of vital importance from the viewpoint of environmental protection to treat contaminated aqueous media such as underground water, soil and the surrounding gaseous phase by removing and degrading the chlorinated organic compounds contained therein and, in fact, efforts have been paid to develop technologies necessary for such treatment operations.
While known technologies for environmental protection include adsorption using active carbon and degradation by heat or light, they are not necessarily viable and feasible in terms of cost and operability.
Meanwhile, microorganisms that degrade chlorinated organic compounds such as TCE that are normally stable in the environment have been reported and a number of researches have been started to exploit them in practical applications. Some of the advantages of using microorganisms for biodegradation processes are that chlorinated organic compounds can be reduced harmless by appropriately selecting one or more specific microorganisms and using them for biodegradation, that basically no chemicals are required for biodegradation and that the cost of maintenance and labor force can be lessened in biodegradation processes. However, there is a limited number of reports on isolated microorganisms having the ability of degrading chlorinated organic compounds. Known microbial strains that can be used for the biodegradation of TCE include Welchia alkenophila sero 5 (U.S. Pat. No. 4,877,736, ATCC 53570), Welchia alkenophila sero 33 (U.S. Pat. No. 4,877,736, ATCC 53571), Methylocystis sp. strain M (Agric Biol. Chem., 53, 2903 (1989), Biosci. Biotech. Biochem., 56, 486 (1992), ibid 56, 736 (1992)), Methylosinus trichosporium OB3b (Am. Chem. Soc. Natl. Meet. Dev. Environ. Microbiol., 29, 365 (1989), Appl. Environ. Microbiol., 55, 3155 (1989), Appl. Biochem. Biotechnol., 28, 877 (1991), Japanese Laid-Open Patent Application No. 2-92274, Japanese Patent Application Laid-Open No. 3-292970), Methylomonas sp. MM2 (Appl. Environ. Microbiol., 57, 236 (1991)), Alcaligenes denitrificans ssp. xylosoxidans JE75 (Arch. Microbiol., 154, 410 (1990)), Alcaligenes eutrophus JMP134 (Appl. Environ. Microbiol., 56, 1179 (1990)), Alcaligenes eutrophus FERM-13761 (Japanese Patent Application Laid-Open No. 7-123976), Pseudomonas aeruginosa JI104 (Japanese Patent Application Laid-Open No. 7-236895), Mycobacterium vaccae JOB5 (J. Gen. Microbiol., 82, 163 (1974), Appl. Environ. Microbiol., 55, 2960, (1989), ATCC 29678), Pseudomonas putida BH (J. of Japan Sewage Work Assoc. (Gesuido Kyokai-shi), 24, 27 (1987), Pseudomonas sp. strain G4 (Appl. Environ. Microbiol., 52, 383, (1986), ibid 53, 949 (1987), ibid 54, 951 (1989), ibid 56, 279 (1990), ibid 57, 193 (1991), U.S. Pat. No. 4,925,802, ATCC 53617, this strain was first classified as Pseudomonas cepacia but later changed to Pseudomonas sp.), Pseudomonas mendocina KR-1 (Bio/Technol., 7, 282 (1989), Pseudomonas putida F1 (Appl. Environ. Microbiol., 54, 1703 (1988), ibid 54, 2578 (1988)), Pseudomonas fluorescens PFL12 (Appl. Environ. Microbiol., 54, 2578 (1988)), Pseudomonas putida KWI-9 (Japanese Patent Application Laid-Open No. 6-70753), Pseudomonas cepacia KKO1 (Japanese Patent Application Laid-Open No. 6-22769), Nitrosomonas europaea (Appl. Environ. Microbiol., 56, 1169 (1990)) and Lactobacillus vaginalis sp. nov (Int. J. Syst. Bacteriol., 39, 368 (1989), ATCC 49540).
A problem in practically applying these decomposing bacteria for remedying the environment is that they require chemicals such as aromatic compounds or methane as an induction substance (an inducer).
While aromatic compounds such as phenol and toluene serve excellently as degradation inducers, they are toxic and should not be released into the environment. While methane is also useful as a degradation inducer, it is highly inflammable and hence it is difficult and highly hazardous to discharge methane into the environment in a controlled manner.
Nelson at al. developed a method of using tryptophan, an amino acid, as a degradation inducer for biodegrading chlorinated organic compounds (Japanese Patent Application Laid-Open No. 4-502277). Although this method can avoid the toxicity and danger of the inducer itself to some extent, tryptophan is a very expensive compound, and complicatedness of introducing a specific substance into the environment with control is not still solved. Additionally, adding excess carbon and nitrogen source into the environment is also not preferable from the viewpoint of eutrophication. Furthermore, since such TCE decomposition enzymes are inducible enzymes, the enzymatic activity once induced is usually sustained for from only several hours to a day, requiring another induction after that, and there is a problem that decomposition of TCE is competitively inhibited by the presence of the inducing agents.
Currently, efforts have been made to introduce an plasmid having a DNA fragment encoding oxygenase or hydroxylase as a TCE decomposition enzyme into a host bacterium in order to express the TCE decomposition activity using a harmless inducer or to constitutively express it in the absence of any inducers. Microbial strains having such a DNA fragment include Pseudomonas mendocina KR-1 (Japanese Patent Application Laid-Open No. 2-503866), Pseudomonas putida KWI-9 (Japanese Patent Application Laid-Open No. 6-105691) and Pseudomonas putida BH (Summary of the Third Conference for the Studies on the Contamination of Underground Water and Soil and Preventive Measures (1994)).
However, these recombinant strains have various different problems including the use of highly expensive IPTG (isopropylthiogalactopyranoside) and an insufficient stability of the plasmid in the host microorganism. Additionally, releasing recombinant microorganisms into the environment is under certain regulations considering the public acceptance.
Shields et al. obtained a mutant strain of Pseudomonas sp. strain G4 capable of degrading TCE in the absence of an inducer (phenol or toluene) using a transposon (Appl. Environ. Microbiol., 58, 3977 (1992), PCT Application, International Publication W092/19738).
However, this mutant strain of G4 is not satisfactorily active in TCE degradation and has a problem of instability due to the transposon. Additionally, since the transposon itself contains an antibiotic resistance gene such as kanamycin resistance, there may be a potential hazard of a horizontal transmission to other microorganisms when the mutant strain is released into the environment.
In view of the above identified problems and other problems, the inventors of the present invention conducted a series of mutation experiments on the J1 strain (FERM BP-5102) by means of nitrosoguanidine and succeeded in obtaining a new strain JM1 (FERM BP-5352) capable of degrading volatile chlorinated organic compounds and aromatic compounds without requiring an inducer as disclosed in Japanese Patent Application Laid-Open No. 8-294387).
More specifically, strain JM1 (FERM BP-5352) was obtained by mutagenizing strain JI (FERM BP-5201; National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology) using a chemical, nitrosoguanidine, without genetic manipulations. Strain JM1 does not require the use of an inducer such as phenol, toluene or cresol for degrading volatile chlorinated organic compounds including TCE.
As a microorganism which can degrade chlorinated aliphatic hydrocarbon compounds such as TCE without an inducer, there has been reported Burkholderia (Pseudomonas) cepacia PR1.sub.301 (hereinafter referred to simply as PR1.sub.301) in Environmental Science & Technology, Vol.30, No.6, 1986, pp.2045-2052.
From the viewpoint of treating waste solutions and soil containing TCE, the microorganism to be used for the treatment is required to have not only a TCE degradability but also the ability of growing and maintaining its degradation activity in the poor environment of waste solution or soil.
In the natural soil environment, unlike culture systems in laboratories, the optimization of the growth and degradation activity of a microorganism capable of degrading chlorinated organic compounds is quite difficult due to the various factors, such as temperature, moisture content, pH, oxygen content and other parameters of soil, so that there still exist a number of problems to be solved for successfully recovering the soil from contamination by means of microorganisms capable of degrading contaminants.
Among these parameters, temperature is very important because it directly affects the growth and the degradation activity of the microorganism introduced in the soil. The soil temperature in the Temperate Zone where Japan is located is held substantially constant to about 15.degree. C., which is significantly lower than 25.degree. C., the optimum temperature for the growth and decomposition activity of strain JM1. While strain JM1 can sufficiently grow and express its biodegradation activity around 15.degree. C. in practical applications, the soil temperature near the surface may fall significantly from that level in winter in Japan where the ambient temperature may fall well below zero. In colder regions, such as cryic and frigid as defined by the Soil Taxonomy of the Department of Agriculture of the U.S. Government, the average annual soil temperature is between 0 and 8.degree. C., and between 8 and 15.degree. C. in mecic.
Therefore, there exists a need for a technology in order to fully exploit the excellent properties of strain JM1 for degrading aromatic compounds and/or chlorinated organic compounds without using an inducer, at a temperature typically between 4 and 15.degree. C.